High Inter- and Intra- Diversity of Amino Acid Content and Protein Digestibility Disclosed in Five Cool Season Legume Species with a Growing Market Demand.

Legumes have been sought as alternative protein sources to ensure food security and environmental sustainability. Characterizing their protein content and quality, including in underutilized grain legumes, e.g., grass pea, gives value to the legumes' underexplored variability. To fill the gap of knowledge in legumes' protein quality, for the first time, five extensive collections of cool season grain legumes were cropped under the same environmental conditions and further analyzed. Multivariate analysis showed the existent intra- and inter-species variability. The legume species with the highest protein content, grass pea, Lathyrus sativus (LS), was not the one with the overall highest individual amino acids content and in vitro protein digestibility. With these last characteristics lentil, Lens culinaris (LC), was highlighted. The highest average values of arginine (Arg), glutamic acid (Glu), and threonine (Thr) were found in LS and Vicia faba (VF). Cicer arietinum (CA) stood out as the species with the highest values of Thr and methionine (Met). Regarding the in vitro protein digestibility (IVPD), LC, followed by Pisum sativum (PS) and LS, were the legume species with the highest values. Ultimately, this study bought to the fore legume species that are not commonly used in western diets but have high adaptability to the European agricultural systems.

Definition of chemical markers for Hancornia speciosa Gomes by chemometric analysis based on the chemical composition of extracts, their vasorelaxant effect and α-glucosidase inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Hancornia speciosa Gomes (Apocynaceae) is a tree found in the Brazilian savannah, traditionally used to treat several diseases, including diabetes and hypertension. The anti-hypertensive activity of H. speciosa leaves (HSL) has been demonstrated in different models and is credited to the vasodilator effect and ACE (angiotensin-converting enzyme) inhibition. The hypoglycemic effect of HSL has been also reported. AIM OF THE STUDY: To establish correlations between the biological activities elicited by H. speciosa extracts and the contents of their major compounds, aiming to define chemical markers related to the potential antihypertensive and antidiabetic effects of the species. Additionally, it aimed to isolate and characterize the chemical structure of a marker related to the α-glucosidase inhibitory effect. MATERIALS AND METHODS: Extracts of a single batch of H. speciosa leaves were prepared by extraction with distinct solvents (ethanol/water in different proportions; methanol/ethyl acetate), employing percolation or static maceration as extraction techniques, at different time intervals. The contents of chlorogenic acid, rutin and FlavHS (a tri-O-glycoside of quercetin) were quantified by a developed and validated HPLC-PDA method. Bornesitol was determined by HPLC-PDA after derivatization with tosyl chloride, whereas total flavonoids were measured spectrophotometrically. Identification of other constituents in the extracts was performed by UPLC-DAD-ESI-MS/MS analysis. The vasorelaxant activity was assayed in rat aortic rings precontracted with phenylephrine, and α-glucosidase inhibition was tested in vitro. Principal component analysis (PCA) was employed to evaluate the contribution of each marker to the biological responses. Isolation of compound 1 was carried out by column chromatography and structure characterization was accomplished by NMR and UPLC-DAD-ESI-MS/MS analyses. RESULTS: The contents of the chemical markers (mean ± s.d. % w/w) varied significantly among the extracts, including total flavonoids (2.68 ± 0.14 to 5.28 ± 0.29), bornesitol (5.11 ± 0.26 to 7.75 ± 0.78), rutin (1.46 ± 0.06 to 1.97 ± 0.02), FlavHS (0.72 ± 0.05 to 0.94 ± 0.14) and chlorogenic acid (0.67 ± 0.09 to 0.91 ± 0.02). All extracts elicited vasorelaxant effect (pIC50 between 4.97 ± 0.22 to 6.48 ± 0.10) and α-glucosidase inhibition (pIC50 between 3.49 ± 0.21 to 4.03 ± 0.10). PCA disclosed positive correlations between the vasorelaxant effect and the contents of chlorogenic acid, rutin, total flavonoids, and FlavHS, whereas a negative correlation was found with bornesitol concentration. No significant correlation between α-glucosidase inhibition and the contents of the above-mentioned compounds was found. On the other hand, PCA carried out with the areas of the ten major peaks from the chromatograms disclosed positive correlations between a peak ascribed to co-eluted triterpenes and α-glucosidase inhibition. A triterpene was isolated and identified as 3-O-ß-(3'-R-hydroxy)-hexadecanoil-lupeol. CONCLUSION: According to PCA results, the vasorelaxant activity of H. speciosa extracts is related to flavonoids and chlorogenic acid, whereas the α-glucosidase inhibition is associated with lipophilic compounds, including esters of lupeol like 3-O-ß-(3'-R-hydroxy)-hexadecanoil-lupeol, described for the first time for the species. These compounds can be selected as chemical markers for the quality control of H. speciosa plant drug and derived extracts.

A Single Dose of Marine Chlorella vulgaris Increases Plasma Concentrations of Lutein, ß-Carotene and Zeaxanthin in Healthy Male Volunteers.

The beneficial health effects of Chlorella vulgaris have been associated with the presence of several nutrients and antioxidants, including carotenoids. However, the in vivo bioavailability of Chlorella is still poorly evaluated. In this work, a human intervention study was conducted in 11 healthy men to evaluate the bioavailability of carotenoids within 3 days after the intake of a single dose (6 g) of dried marine Chlorella vulgaris containing lutein (7.08 mg), ß-carotene (1.88 mg) and zeaxanthin (1.47 mg). Subjects were instructed to follow a low carotenoid diet during the experimental phase, starting 1 week earlier. On the day of the experiment, dried microalgae formulated in vegetarian hard capsules were ingested, and blood samples were collected up to 72 h for the analysis of plasma carotenoids concentration by high-performance liquid chromatography with diode-array detection. For all carotenoids, the estimated AUC and Cmax values were significantly different from zero (p < 0.05), indicating that a single dose of marine Chlorella vulgaris increased plasma concentrations of lutein (Cmin-corrected AUC = 1002 µg·h/L, Cmax = 20.4 µg/L), ß-carotene (AUC = 1302 µg·h/L, Cmax = 34.9 µg/L) and zeaxanthin (AUC = 122.2 µg·h/L, Cmax = 3.4 µg/L). The bioavailability of other compounds, namely, polyunsaturated fatty acids and trace elements, was also assessed post-prandial for the first time, showing that linoleic acid, docosahexaenoic acid and iodine were absorbed after microalgae intake. These findings support the use of Chlorella vulgaris as a source of carotenoids, PUFA and essential trace elements with associated health benefits.

Projection to latent correlative structures, a dimension reduction strategy for spectral-based classification.

Latent variables are used in chemometrics to reduce the dimension of the data. It is a crucial step with spectroscopic data where the number of explanatory variables can be very high. Principal component analysis (PCA) and partial least squares (PLS) are the most common. However, the resulting latent variables are mathematical constructs that do not always have a physicochemical interpretation. A new data reduction strategy, named projection to latent correlative structures (PLCS), is introduced in this manuscript. This approach requires a set of model spectra that will be used as references. Each latent variable is the relative similarity of a given spectrum to a pair of reference spectra. The latent structure is obtained using every possible combination of reference pairing. The approach has been validated using more than 500 FTIR-ATR spectra from cool-season culinary grain legumes assembled from germplasm banks and breeders' working collections. PLCS has been combined with soft discriminant analysis to detect outliers that could be particularly suitable for a deeper analysis.

Identification of functional compounds in baru (Dipteryx alata Vog.) nuts: Nutritional value, volatile and phenolic composition, antioxidant activity and antiproliferative effect.

This work aimed to contribute to the nutritional and functional characterization of roasted baru nuts, a seed widely consumed and produced in Brazil. Baru nut was characterized in terms of its nutritional value and volatile composition (SPME-GC-MS analysis). The ultrasound assisted extraction was used to extract free and bound phenolic compounds that were identified by LC-DAD-ESI-MS/MS method. Bioactivity assays were carried out to evaluate the antioxidant activity (ORAC and HOSC assay) and anticancer effect (inhibition of HT29 cell growth and targeting of cancer stemness) of baru nut extracts and phenolic compounds. Results showed that baru is a good source of protein and monounsaturated fatty acids, specifically oleic acid (47.20 g/100 g). The predominant volatile compounds are hexanal (71.18%) and 2,5-dimethyl-pyrazine (9.43%). The main phenolic compounds identified were gallic acid and its derivatives, such as gallic acid esters and gallotannins. Among all, gallic acid and methyl gallate seemed to be the main compounds responsible for the high antioxidant activity. The antiproliferative effect evaluated of baru extracts in HT29 cell line showed ability to impair cell growth in both monolayer and spheroid cultures and to reduce ALDH+ population. These results supply new information about the functional compounds presents in baru nut, which are important sources of natural antioxidants and antiproliferative compounds.

Evaluation of the Wound Healing Properties of Hancornia speciosa Leaves.

The leaves of Hancornia speciosa Gomes (Apocynaceae), a medicinal species found in the Brazilian cerrado biome, are traditionally used to treat wounds and inflammatory disorders. The goal of the present study was to investigate the in vitro wound healing properties of ethanolic extract of H. speciosa leaves and its isolated compounds, using the scratch assay, and to evaluate their effects on the release of the pro-inflammatory cytokine tumor necrosis factor (TNF-α) by lipopolysaccharide (LPS)-stimulated human acute monocytic (THP-1) cells. H. speciosa ethanolic extract significantly increased (42.8% ± 5.4 at 25 µg/mL) cell migration and proliferation of fibroblasts compared with control cells, as well as the isolated compounds bornesitol (80.8% ± 5.1) and quinic acid (69.1% ± 6.2), both assayed at 50 µM. TNF-α release by LPS-stimulated THP-1 cells was significantly reduced by the ethanolic extract (62.9% ± 8.2, i.e. 1791.1 ± 394.7 pg/mL) at 10 µg/mL, bornesitol (48.9% ± 0.9, i.e. 2461.6 ± 43.1 pg/mL) at 50 µM, and quinic acid (90.2% ± 3.4, i.e. 473.5 ± 164.4 pg/mL) and rutin (82.4% ± 5.6, i.e. 847.0 ± 271.8 pg/mL) at 10 µM. These results provided evidences to support the traditional use of H. speciosa leaves to treat wounds and inflammatory disorders.

Hancornia speciosa Gomes (Apocynaceae) as a potential anti-diabetic drug.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Hancornia speciosa Gomes are traditionally used to treat diabetes in Brazil. The aim of the study is to evaluate the potential anti-diabetic effect of Hancornia speciosa extract and derived fractions. MATERIALS AND METHODS: The ethanolic extract from Hancornia speciosa leaves and chromatographic fractions thereof were evaluated on α-glucosidase assay, on hyperglycemic effect and glucose uptake. The chemical composition of the extract and its most active fraction was investigated by ESI-LC-MS. RESULTS: The ethanolic extract and derived fractions inhibited α-glucosidase in vitro. However, only the crude extract and the dichloromethane fraction inhibited the hyperglycemic effect induced by starch or glucose. Both the extract and dichloromethane fraction were also able to increase glucose uptake in adipocytes. Bornesitol, quinic acid, and chorogenic acid were identified in the extract, along with flavonoid glycosides, whereas the dichloromethane fraction is majorly composed by esters of lupeol and/or α/ß-amirin. CONCLUSIONS: Hancornia speciosa has a potential anti-diabetic effect through a mechanism dependent on inhibition of α-glucosidase and increase on glucose uptake. These results give support to the use on traditional medicine of this medicinal plant.

Seasonal variation on the contents of coumarin and kaurane-type diterpenes in Mikania laevigata and M. glomerata leaves under different shade levels.

Coumarin (1) and kaurane-type diterpenes are considered the bioactive constituents of Mikania glomerata and M. laevigata, used in Brazil to treat respiratory affective disorders. The seasonal variation of 1, ortho-coumaric acid (2), benzoylgrandifloric acid (3), cinnamoylgrandifloric acid (4), and kaurenoic acid (5) in leaves of both species, cultivated in full sunlight and under shade levels of 40 and 80%, was quantified by HPLC. Compound 2 was detected solely in M. laevigata in concentrations below the limit of quantification. Coumarin was not found in M. glomerata, whereas its concentration reached 0.94±0.24% (w/w) in M. laevigata farmed in summer under 80% shading. Both Mikania species produced higher amounts of kaurane diterpenes when cultivated in plenty of sunlight. Hence, maximum contents of 1 are reached in M. laevigata cultivated under high shading, but with reduced concentrations of 3-5. Conversely, M. glomerata should be cultivated under full sunlight and harvested in winter for highest concentrations of kaurane-type diterpenes.

Development and validation of an HPLC-DAD method for quantification of bornesitol in extracts from Hancornia speciosa leaves after derivatization with p-toluenesulfonyl chloride.

Cylitol L-(+)-bornesitol is regarded as a bioactive constituent from Hancornia speciosa leaves, a plant species traditionally used in Brazil to treat diabetes and hypertension. We report a simple HPLC-DAD method for the quantification of bornesitol in extracts from H. speciosa leaves, after derivatization with p-toluenesulfonyl chloride, using pentaerythritol as internal standard. A gradient of methanol, acetonitrile and water was employed for elution on an ODS column and detection was set up at a wavelength of 230 nm. The method was selective and linear over the range 60.4-302.0 µg/ml with r² of 0.9981, and showed satisfactory precision for intra-day (RSD=2.37%) and inter-day (RSD=3.17%) assays. The recovery varied between 92.3% and 99.9% and the limits of quantification and detection were respectively 5.00 and 1.67 µg/ml. The method was applied to quantify bornesitol in extracts from H. speciosa leaves of different specimens.

Development and validation of an RP-HPLC method for quantification of cinnamic acid derivatives and kaurane-type diterpenes in Mikania laevigata and Mikania glomerata.

MIKANIA GLOMERATA and MIKANIA LAEVIGATA (Asteraceae) are medicinal plants popularly named 'guaco' in Brazil. The leaves of both species are used to treat respiratory diseases, with coumarin (CO) and kaurane-type diterpenes being regarded as the bioactive constituents. A new and simple RP-HPLC method was developed and validated for the simultaneous quantification of CO, O-coumaric (OC), benzoylgrandifloric (BA), cinnamoylgrandifloric (CA) and kaurenoic (KA) acids in the species. Optimal separation was achieved with an alternating gradient elution of methanol and acetonitrile and detection was carried out by DAD at three different wavelengths: 210 nm for CO, OC, KA; 230 nm for BA; and 270 nm for CA. The extracts showed good stability during 42 hours under normal laboratory conditions (temperature of 23 +/- 2 degrees C). The standard curves were linear over the range 0.5 - 5.0 microg (CO), 0.25 - 4.0 microg (OC), 1.0 - 8.0 microg (BA), 0.5 - 3.0 microg (CA) and 0.8 - 12.0 microg (KA), with R(2) > 0.999 for all compounds. The method showed good precision for intra-day (RSD < 4.6 %) and inter-day assays (RSD < 4.4 %). The recovery was between 99.9 and 105.3 %, except for CO and OC in M. glomerata (73.2 - 91.6 % and 86.3 - 117.4 %, respectively). The limits of quantification and detection were in the range of 0.025 - 0.800 microg and 0.007 - 0.240 microg. The method was tested for new and old columns, temperature variation (26 and 28 degrees C) and by different operators in the same laboratory. The method was successfully applied to samples of both species.